Amniotic fluid derived mesenchymal stem cells (AFS cells) have been considered as an applicable treatment of stem cell therapy in regenerative medicine. AFS cells have similar differentiation ability to embryonic stem cells with less ethical controversy. For applying in regeneration of injured and damaged tissues there are still considerable huddles which need to be overcome. One of the crucial problems is how sustains or even amplifies their multi–potent ability till administration in damaged tissue/organ sites. The 2–D adherent culture method provides different environment compared to those of in vivo. 3–D culture method is an alternative manner to culture cells in in vivo–like cell culture condition. Hanging drop is one of simple and effective 3–D culture systems. Here canine AFS cells were cultured in the hanging drops in which cells form spheroids with micro–networks that allow exchanging cellular signalings similar to in vivo. Canine AFS cells were aggregated in tightly spaced spheres in hanging drops supplemented with 20% FBS. In the study of cellular senescence, AFS cells successfully formed and maintained spheroids regardless of their passages. In addition, the multipotent differentiation capacity of AFS cells in shape of 3–D spheroids was enhanced based on MSC differentiation assay after hanging drop culture. Noticeably, the expression levels of cartilage oligomeric matrix protein (COMP) and aggrecan (AGG) were significantly increased in 3–D spheroid cultured AFS cells compared to 2–D adherent cultured controls. Therefore, induction of spheroid formation by hanging drops is a useful method to provide 3–D in vivo–like culture condition without changes of AFS cells differentiation capacity especially to induce chondrogenic differentiation for regenerating damaged cartilages.